In 1983, the etiological cause of AIDS was determined to be the human immunodeficiency virus (HIV). In 1985, it was reported that the synthetic nucleoside 3′-azido-3′-deoxythymidine (Zidovudine, AZT, ZDV) inhibits the replication of human immunodeficiency virus by inhibiting in its 5′-triphosphate form the HIV-1 reverse transcriptase (HIV-RT). HIV-RT is active early in the viral replication cycle and is necessary for continued viral replication. Currently, a total eight synthetic nucleosides have been approved by the US FDA. These are: AZT (mentioned above), 2′,3′-dideoxyinosine (Videx, DDI), 2′,3′-dideoxycytidine (DDC), 2′,3′-dideoxy-2′,3′-didehydrothymidine (stavudine, D4T), cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (emtricitabine, FTC), (−)-cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (Lamivudine, 3TC), (1S,4R)-4-[2-amino-6-(cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-methanol succinate (abacavir, ABC), and the acyclic nucleotide 9-[(R)-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate (tenofovir-DF, TDF). All nucleoside reverse transcriptase inhibitors (NRTI) require phosphorylation to their triphosphate (TP) forms, while metabolic activation of tenofovir requires phosphorylation to its 12 diphosphate (tenofovir-DP). Such so-called NRTI mimic natural nucleosides in the cell. After cellular phosphorylation to the 5′-triphosphate by cellular kinases, these synthetic nucleosides can be incorporated into a growing strand of viral DNA, causing chain termination due to the absence of the 3′-hydroxyl group found in natural nucleosides that are used in the DNA chain elongation reaction catalyzed by HIV-RT. NRTI therapies in HIV treatment are reviewed in Schinazi et al., Antiviral Research 71:322-334 (2006)).
HIV shows high genetic variability in part as a result of its fast viral replication cycle coupled with the high mutation rate of and active recombinogenic characteristics of HIV-RT, especially during viral replication in single cells co-infected by multiple different strains of HIV. Drug-resistant variants of HIV can emerge after treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication, and most typically in the case of HIV, reverse transcriptase, protease, or DNA polymerase. NRTI treatment of HIV-1 infected individuals often leads to the emergence of mutations in the reverse transcriptase (RT). Less frequently seen are codon insertions or deletions, either which add or subtract three nucleotides and leave other codons in the correct coding frame. Codon insertions (ins) and deletions (del) have been associated with multi-drug resistance (MDR) in clinical samples obtained from HIV-1 infected individuals treated with antiretroviral agents (67del, 69del, 69ins, 70del).
The β3-β4 hairpin loop of the finger domain of RT is thought to be directly involved in the interaction of the enzyme with its substrates (the template-primer complex and the dNTP) (Tamalet et al., Virol. 270:310-316 (2000)). Genetic rearrangements in the β3-β4 loop have been found in patients extensively treated with anti-HIV drugs and experiencing therapeutic failure (Tamalet, supra; Winters et al., J. Virol. 74(22):10707-10713 (2000)).
The efficacy of a drug against HIV infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and in particular a third, antiviral compound that induces a different mutation from that caused by the principle drug. Alternatively, the pharmacokinetics, biodistribution, or other parameter of the drug can be altered by such combination or alternation therapy, although this is not recommended for HIV infections. In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous pressures on the virus. One cannot predict, however, what mutations will be induced in the HIV-1 genome by a given new drug, whether the mutation is permanent or transient, or how an infected cell with a mutated HIV-1 sequence will respond to therapy with other agents in combination or alternation. This is exacerbated by the fact that there is a paucity of data on the kinetics of drug resistance in long-term cell cultures treated with modern antiretroviral agents.
HIV-1 variants resistant to AZT, DDI, 3TC, D4T, DDC, ABC or TDF have been isolated from patients receiving long term monotherapy with these drugs (Larder et al., Science 1989; 243:1731-4; St. Clair et al., Science 1991; 253:1557-9; and Fitzgibbon et al., Antimicrob. Agents Chemother. 1992; 36:153-7; Schinazi, et al., Intl. Antiviral News 2000; 8:65-92). Mounting clinical evidence indicates that AZT and 3TC resistance is a predictor of poor clinical outcome in both children and adults. The rapid development of HIV-1 resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) has also been reported both in cell culture and in human clinical trials (Nunberg et al. J. Virol. 1991; 65(9):4887-92; Richman et al., Proc Natl Acad Sci (USA) 1991; 88:11241-5; Mellors et al., Mol. Pharm. 1992; 41:446-51; Richman D D and the ACTG 164/168 Study Team. Second International HIV-1 Drug Resistance Workshop. (Noordwijk, the Netherlands. 1993); and Saag et al., N Engl J Med 1993; 329:1065-1072). In the case of the NNRTI L'697,661, drug-resistant HIV-1 emerged within 2-6 weeks of initiating therapy in association with the return of viremia to pretreatment levels. (Saag et al., supra). Breakthrough viremia associated with the appearance of drug-resistant strains has also been noted with other classes of HIV-1 inhibitors, including protease, fusion and integrase inhibitors. This experience has led to the realization that the potential for HIV-1 drug resistance must be assessed early on in the preclinical evaluation of all new therapies for HIV-1.
The emergence of resistant HIV strains during viral therapy has presented a major challenge to delay, prevent or attenuate the onset of resistance. Common resistance mutations, including thymidine associated mutations (TAM), K65R and M184V are problematic in HIV drug development. Mutations observed to emerge following exposure to various NRTI are summarized in Schinazi et al., supra, 2006 (see Table 1). Novel NRTI are under pre-clinical development that are good substrates for cellular kinases, have high bioavailability (especially oral), reduced toxicity and significant levels of activity against the commonly found NRTI-resistant HIV-1 mutants, such as D67N, K70R, T215Y, K219Q, K65R and M184V (Chu et al., J. Med. Chem. 48:3949-3952 (2005)).
2′,3′-Dideoxy-2′,3′-didehydro-5-fluoro-cytidine (D4FC, DFC; dexelvucitabine) is a known NRTI compound (see, e.g., EP 0 409 227 A2, U.S. Pat. Nos. 5,703,058 and 5,905,070). Treatment with β-L-D4FC rapidly selects for a mutation at codon 184 (methionine to valine) of the reverse transcriptase region of the virus, resulting in a high level of resistance to 3TC and FTC. β-D-D4FC, in contrast, is not significantly cross-resistant to AZT, DDC, DDI, D4T, 3TC, (−)-FTC or β-L-D4FC. β-D-D4FC treatment selects for HIV-1 variants having mutations at codons I63L, K65R, K70N, K70E, or R172K of the HIV-RT region of the virus (see also Hammond et al., Antimicrob. Agents Chemother. 49(9):3930-3932 (2005)). Thus, β-D-D4FC can be used generally as salvage therapy for any HIV-infected individual that exhibits resistance to other anti-HIV agents whose drug resistance patterns correlate with mutations at codons different from those selected by β-D-D4FC treatment. Based on this, methods for treating HIV have been reported that involve administering β-D-D4FC or its pharmaceutically acceptable salt or prodrug in combination or alternation with a drug that selects for variants having one or more mutations in HIV-1 at a location other than codons I63L, K65R, K70N, K70E, or R172K (U.S. Pat. No. 7,115,584, and Hammond et al.).
Current treatments for HIV infection are most often those referred to as “highly active antiretroviral therapy” or HAART and involve administering combinations (“cocktails”) comprising at least three drugs—two NRTI in combination with either a protease inhibitor or a NNRTI. Results of studies on the emergence of drug resistance and correlations between antiviral drugs and mutation patterns present in selected HIV variant genes are useful in directing resistance testing of viruses from HIV-infected individuals treated with antiviral agents such as NRTI and in choosing combinations of nucleoside analogs for treatment and prevention of drug resistant HIV. Characterization of these mutations is key in determining potential cross-resistance and in HIV treatment management. It is thus desirable to understand more about NRTI resistance patterns and how they correlate with HIV genotypes and mutations in essential HIV genes, such as HIV-RT.